The Establishment Of The Technical Platform Of CDNA Microarray Of Tumor Metastasis Associated Genes And Its Application

Background: Microarray technology is a new technology that developed by the implement and completion of human genome project. It is key new technology that has draw attention of many scientist and company. It has found a lot of application prospect in the gene expression analysis, high throughput drug screening, clinical diagnosis, forensic use etc. In order to promote the application of the cDNA microarray in the tumor diagnosis and the prediction of prognosis or molecular phase of tumor, we have established a technological platform for the preparation of cDNA microarray, slide processing , reverse trancription labeling method and the optimization of hybridization condition.1. The establishment of technical platform of cDNA microarray of tumor metastasis associated genes.(1) The selection of target genes and the optimization of fabrication of cDNA microarray.With the completion of human genome project(HGP) and the establishment of public gene information database (such as Genebank, DDBJ, EMBL) it made it possible for the scientist to make use of the large amount of genes information. In order to prepare a microarray focusing on the tumor classification and in order to fabricate a metastasis associated gene microarray that aimed at the clinical application for the predication of prognosis or make clinical classification and molecular phase and anti- matastasis drug target screening, we have selected the target genes that were related to the metastasis of tumor from the three world gene databases and the differential expression genes revealed by literaure of microarray. Firstly, total of 447 genes and ESTs were selected, and then the IMAGE ID were identified in order to purchase the Clones from the IMAGE Consortium sources—one of the three sources of gene clones—research Genetics(Now merged into Invitrogen Life Technoligies.). The target genes include oncogenes, tumor suppressor genes, growth factor and related receptors, signal transduction molecules and protein synthesis molecules and someESTs. A mini cDNA clone banks were established for the preparation of the tumor metastasis associated microarray. All the cDNA clones were constructed into the vector that has the univerasal M13 forward and reverse sequence. A series of optimization have been done for the preparation of microarray such as the extraction of plasmids from the cloned bacteria, PCR amplication of the target genes, purification of the PCR products, the comparison of spot solution, the dosage of Ultro-violet crosslink and the selection of different kinds of slides. The results showed that the extraction of plasmids with Edgebiosystem 96-well plate Plasmids extraction kits can acquire good quality plasmids. A 90% success amplication rate target genes can be reached with M13 vector specific primers. A good spot effects obtained by resuspending the PCR products with arrayit spot solution . The polylysine coated glass slides and aninosalinate glass slides of cell associates all can produced uniform spot images.(2) Optimization of reverse transcription labeling of cDNA first strand for the use of microarray hybridizationThere are at least four methods for the labeling of the first strand cDNA,that is direct labelling with cy-dye labelled dUTP or dCTP,indirect labeling with aminoallyl-dUTP,Genesphere 3DNA indirect labeling and in vitro transcription indirect labeling method. We chose the first two mehtod that is relative cheap for optimization in our study. The results showed that the indirect method with aminoallyl dUTP was significantly superior to the direct labeling method. We then do some optimization on indirect method. We have compared three RNA extraction reagents, two reverse transcrtiption primers, different time of reverse transcription, three dosages of RNA for labbelling and two kinds of reagents for blocking coupling of cy dye with cDNA, different hybridization temperature,and the washing buffer were also compared. The results showed that the Trizol reagents of the three company were all good enough to extract the wholesome RNA with relative little contaminiation of protein. The reverse transcription primed with random hexamer were significantly superior to poly-dT primers(12-18 mers) .The hybridization signal of overnight transcription were markedly more intense than that of the 2-3h reverse transcription with relative low background and high signal-noise ratio. To block the coupling with sodium acetate is better than that with 4M hydroxyamine. The hybridization at 42 °C overnight is better than that of 65 °C with relative low background and high signal /noise.(3) The establishment of gene annotation, the Quantarray analysis ofmicroarray hybridization results,and the comparison of three normalization methodTo establish the gene annotation, in the Quantarray Software, find the FillgenelD program and fill all the genes information into the table to establish the genes annotation specific for our metastasis assocaited genes microarray. Then it is can be used in the Quantarray of microarray results so that the intensity of every spot in the microarray were correspond to the gene information. The Quantarray method was also eastablished. In comparison of the the three normalization method , for the same Quantarray data, the three method has the identical results. But when the houskeeping genes changes are predicted, it is better to use total or median normalization method.(4) The validation of stability, reproducibility and cutoff values of the cDNA microarrayTo validate the stability of the microarray after spoting, two time point of crosslink were compared either immediately after the dryness of the microarray or before postprocessing. The expectancy of preserving either before or after post processing were also compared. It was found that the immobilization effects of target genes crosslinked immediately after spoting were better than before post processing. The expectancy of the target genes immobilization is at least 6 monthes. The fluorescence intensity are stable for at least 8 monthes after hybridization on condition that the microarray was kept at dark.The reproducibility of hybridizations intra microarray and inter microarray were also evaluated . Reproducible results were obtained either intra microarray or inter microarray. The cutoff values to determine differential expression genes was measured with self-against-self hybridization method. Four tumour cell lines with each having two replicates were used for the measurement of cutoff values. The ratio of upper 95% confidence limit for the differential expression genes is 1.66, and the lower 95% confidence limit was 0.61. The international cutoff values were 2 for up-pregulated genes and 0.5 for down-regulated genes. Therefore, we have more confidence to use the 2 or 0.5 as cutoff value for determining the differential expression genes.2.The application of cDNA microarray of tumor metastasis associated genes in the gene expression analysis and screening of the target genes of drug(1) The expression of tumour metastasis associated genes in the rectal cancer and its use in classifications of tumor stage.The differntial expression genes between the tumour tissue and paired normal tissue of the same patients in 21 colerectal cancer were analyzed with the cDNA microarray deveolped as stated above. 24 genes were found regularly up-regulated and 14 genes down -regulated which included known genes and ESTs. The Claufavour Version 5.0 software was used for the calssification of rectal cancer patients. The results showed that the patients can be divided by two group,one is the clinical stage that belonged to II III IV ,the other is belong to I .The principal component analysis results showed that all the genes can be divided into 7 major groups. In these groups there is 17genes that are positive correlated with the principal component 1 of 7(r>0.45),and there are 9 genes that negative correlated with component3 of the 7(r<-0.45). The differntial expression genes were also compared between cell lines with high metastasis potency (LoVo) and low metastsasis potency (SW480). 36 genes were found up-regulated and 17 genes were found down-regulated in cell lines with high metastasis potency compared with low metastasis potency.(2) The application cDNA microarray of metastasis associated genes in the identification of differential expression genes of oesophagous squamous carcinoma18 pairs of tumor tissues and normal tissues were also analysed with the cDNA microarray of tumor metastasis associated genes with the consent of the patients. The results showed that there were 42 genes up-regulated and 36 genes down -regulated in tumor tissues compared to paired normal tissues. The differntial expression genes included:oncogenes and tumor suppressor genes ,matrix metaloproteinase , adhesion molecules, angiogenesis related growth factor, signal pathway molecules and apoptosis associated genes. These genes are the potential molecular markers for the oesphageal squamous carcinoma.(3) The use of cDNA microarray of tumor metastasis associated genes in the screening of target genes of aspirinAspirin is a non-steriods anti-immflamatory drugs that has been used in the chmoprecvantion and anti-metastasis of colorectal cancer. We here tried to found the target genes of aspirin in the treatment of ovarain ccancer. Ovarian cell lines 3AO was treated with 1.2mMol/L aspirin for 16h and 24 h respectively. The RNA was extracted with Trizol reagents and labelled with animoallyl indirect labeling method and then coupled to monofunctional cy3 or cy5 dye ester and the fluorescence labelled cDNA were applied to microarray for the detection of gene expression. THEresults showed that the hopothetical protein,RAB2,pyrovate kinase muscle and EST were up-regulated and the PECAM1,PAI2,ADAMTSA1,ECGF1,VCAMS, FER were down- regulated which were adhesion and signal tranducrion molecules. After treatment for 48 h,more genes were affected by aspirin, within which beta-2 microglubulin,RAB2, ribsomal proteinl9,HDACl,HSPAlAl,MTAl,NM23-H6 AND some ESTS.And collegen,typeIII alpha,spectrin, alpha,non-erythrocytic alpha-foldrin). These genes may be the new target genes for anti-matastasis effects in ovearian carcinoma.(4) The use of cDNA microarray of tumor metastasis associated genes in the screening of the target genes of cyclooxygnase-2 (COX-2) selective inhibitor.NS398 is a selective COX-2 inhibitor and more and more attention have been focused on its anti-cancer effects . To found the possible target genes of of its anticancer effects,100 P mol/L NS398 were used to treat the colorectal cancer cell lines LoVo which has high potential of metastsis . The results showed that after treatment for 24h with NS398, nine genes were up-regulated and 8 genes were down-regulated. Among the up-regulated genes, there were 5genes continually up-regulated until 48h after treatment which were all ESTs. The down-regulated genes included Iipocalin2(oncogene 24p3,LCN2), intercellular adhesion molecules 5,telencephalin(ICAM5) ,v-jun avian sarcoma virusd 17 oncogene homolog(JUN),V-rel avian reticuloendothelial viral oncogene homolog(REL). After treatment for 48h ,more genes are affected by NS398. There were 31 genes up-regulated and 14 genes down-regulated. In the 31 up-regulated genes,IGFBP-5,APAF, LAMP2,CLCA2,1 aminin gamma3,HOXAl,WNT2, N-cadherin, PAI2, TIMP were all the suppressor of metastasis of tumor. In the down regulated genes ,TAC1,APS,BRCA2 were never reported as the target genes of NS398. Further study will be needed to make clear its significance. CDNA microarray was a useful tools for the screening of the target genes.3. ConclusionWe have established a technological platform of cDNA microarray and found its application in the gene expression and clinical classification and for the target gene screening of drugs.